1. Field of the Invention
The present invention relates to a method of producing saccharides containing glucose as the major constituent by an enzymatic saccharification in which cellulose or hemicellulose in a biomass is enzymatically degraded. Particularly, the present invention relates to a method of producing saccharides containing glucose as the major constituent capable of increasing glucose production by an efficient enzymatic saccharification with a small amount of enzyme.
Priority is claimed on Japanese Patent Application No. 2010-293275, filed Dec. 28, 2010, the content of which is incorporated herein by reference.
2. Description of Related Art
Nowadays, research and development of techniques for producing bioethanol from a cellulosic biomass are conducted all over the world. Woods, grasses, residuals of agricultural products, used papers, paper sludges, cotton fibers, or the like, are examples of the cellulosic biomass. More specifically, construction waste, wood wastes, straws, bagasse (the fibrous matter that remains after sugarcane or sorghum stalks are crushed), corn stover, or the like can be named as examples of the cellulosic biomass.
The concentrated and diluted sulfuric acid methods, and the enzymatic method have been developed as methods of producing bioethanol, in which saccharides produced from the cellulosic biomass are fermented to produce ethanol. In recent years, the enzymatic method has drawn particular attention.
In the enzymatic method, cellulose and hemicellulose in the biomass are degraded with an enzyme to produce saccharides. Then, the produced saccharides are fermented to ethanol by fermenting microorganisms, such as yeast.
Cellulose is a simple polysaccharide that is formed by dehydration condensation of glucose. Thus, when the cellulose is hydrolyzed (enzymatic degradation), glucose is produced.
Hemicellulose is a complex polysaccharide that is formed by dehydration condensation of glucose, xylose, mannose, and the like. Thus, when the hemicellulose is hydrolyzed (enzymatic degradation), glucose, xylose, mannose and the like are produced.
A fermentating microorganism is added to a solution containing saccharides obtained by the enzymatic saccharification using cellulose and hemicellulose. Then ethanol is produced by fermentation of the saccharides.
In the conventional enzymatic saccharification of cellulose and hemicellulose, a biomass containing cellulose and/or hemicellulose and a solution containing an enzyme (enzyme solution) are mixed to prepare a mixed solution (slurry). Then, an enzymatic saccharification of the cellulose and/or hemicellulose is performed in a condition where the mixed solution is stirred or shaken. It has been reported that the lower the amount of enzyme used, the lower the amount of sugar produced in the conventional enzymatic saccharification of cellulose and hemicellulose.(see Non-Patent Literature 1: W. Sattler, H. Esterbauer, O. Glatter, W. Steiner, “The Effect of Enzyme Concentration on the Rate of the Hydrolysis of Cellulose” Biotechnology and Bioengineering, Vol. 33, pp. 1221-1234 (1989); Non-Patent Literature 2: Yanpin Lu, Bin Yang, David Gregg, John N. Saddler, Shawn D. Mansfield, “Cellulase Adsorption and an Evalution of Enzyme Recycle During Hydrolysis of Steam-Exploded Softwood Residues” Applied Biochemistry and Biotechnology, Vols. 98-100, 2002; Non-Patent Literature 3: Farzaneh Teymouri, Lizbeth Laureano-Perez, Hasan Alizadeh, Bruce E. Dale, “Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover” Bioresource Technology 96, pp. 2014-2018, 2005; Non-Patent Literature 4: Ming Chen, Liming Xia, Peijian Xue, “Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate” International Biodeterioration & Biodegradation 59 (2007) 85-89).
It is very critical to reduce the amount of the saccharifying enzyme to put the enzymatic saccharification process into practical use, since the saccharifying enzyme is expensive. However, the problem of lowered sugar production associated with reduced enzyme loading has been preventing the reduction of the enzyme loading. However, the reason causing the problem has not been understood, and a way to solve the problem has not been established. Therefore, research and development has been focused mainly on 1) increasing enzymatic activity of the saccharifying enzyme and 2) increasing efficiency of the saccharifying enzyme by modifying the structure of biomass, such as the crystal structures of cellulose.
It has been found that the amount of the saccharifying enzyme can be reduced without a significant ratio of the enzyme being deactivated by optimizing the reaction condition of the biomass including the saccharifying enzyme and cellulose and/or hemicellulose (Patent Literature 1: WO 2011/074479). As explained above, in the conventional method, the enzymatic degrading reaction, in which cellulose and/or hemicellulose are reacted with the saccharifying enzyme, is performed under the condition where the mixed solution is stirred or shaken. Contrary to that, in the method describe in Patent Literature 1, the enzymatic saccharification is performed under a condition where the mixed solution is undisturbed without stirring and shaking. In the method, the mixing solution is undisturbed, or only subjected to intermittent stirring or shaking. In the method described in Patent Literature 1, the amount of the saccharifying enzyme can be reduced without a significant ratio of the enzyme being deactivated.
The reason why the amount of the saccharifying enzyme can be reduced without a significant ratio of the enzyme being deactivated when the mixed solution is not stirred or shaken is explained below.
Conventionally, it has been believed that reaction rate of the enzymatic degradation is improved when the mixed solution is stirred or shaken, since temperature and ingredients of the solution are more evenly distributed in the reaction tank. Therefore, in the conventional enzymatic saccharification, the enzymatic saccharification is performed by stirring the mixed solution (slurry), after preparing the mixed solution (slurry) by mixing a biomass containing cellulose and/or hemicellulose and a solution containing a saccharifying enzyme (enzyme solution). As a result, there are many reports describing case studies investigating a slurry stirring condition and a slurry stirring apparatus. For example, effects of 1) speed of stirring, 2) shape of stirring impeller, 3) structure stirring apparatus, and the like are investigated (see Non-Patent Literature 5: M. Sakata, H. Ooshima, Y Harano, “EFFECTS OF AGITATION ON ENZYMATIC SACCHARIFICATION OF CELLULOSE” Biotechnology Letters, Vol.7, No. 9, pp. 689-694 (1985); Non-Patent Literature 6: Hanna Ingesson, Guido Zacchi, Bin Yang, Ali R. Esteghlalian, John N. Saddler, “The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose” Journal of Biotechnology 88, pp. 177-182 (2001); Non-Patent Literature 7: Henning Jorgensen, Jakob Vibe-Pedersen, Jan Larsen, Claus Felby, “Liquefaction of Lignocellulose at High-Solids Concentrations” Biotechnology and Bioengineering, Vol. 96, No. 5, pp. 862-870, Apr. 1, 2007; and Non-Patent Literature 8: M. Sakurai, Y Takahata, K. Takahashi, “Stirring Operation in Enzymatic Saccharification of Cellulosic Biomass” Chemical Engineering, pp. 68-72, March (2009)).
However, the effect of enzyme concentration has not been paid attention in these reports. It has been known that the saccharifying enzyme, which hydrolyzes cellulose and/or hemicellulose into monosaccharide, is deactivated when it is subjected to a high physical stress due to stirring, shaking, or the like. However, it has not been known that extent of the enzyme deactivation can vary extremely depending on the concentration of the saccharifying enzyme. Here, the inventors found that when the enzyme concentration is high, the deactivation rate of the saccharifying enzyme can be reduced even if the enzyme solution is subjected to a physical stress. Also, when the enzyme concentration is low, deactivation rate of the saccharifying enzyme was extremely high in case where the enzyme solution is subjected to the same physical stress (see FIG. 4).
With an experimental data showing rate of enzyme deactivation is low in a condition where the concentration of the saccharifying enzyme is high, the stirring process has been performed in a condition where the concentration of the enzyme is low in the conventional methods, since the enzyme concentration effect to the physical tolerance of the enzyme has not been known. That leads to a significant deactivation of the enzyme occurs, making it difficult to reduce the amount of the enzyme. In the present specification, deactivation means that an enzyme having a hydrolyzing activity loses the activity. More specifically, it means an enzyme having an activity to hydrolyze cellulose and/or hemicellulose into a monosaccharide loses the activity.
The phenomenon described above is explained using FIG. 4. FIG. 4. is a graph showing a relationship between reaction time (x-axis) and amount of enzyme deactivation (y-axis) during incubation.
In the reaction, four 100 mL-volume enzyme solutions were prepared by dissolving an enzyme to 100 ml of 50 mM acetate buffer, pH of which is adjusted to 5. Enzyme concentration of the first and second enzyme solutions were 0.6 g/L and that of the third and fourth enzyme solutions were 6 g/L. Then, the four enzyme solutions were incubated at 50° C. for indicated period. During the incubation, the first and third enzyme solutions were subjected to stirring density of 73 W/m3, while the second and fourth enzyme solutions were allowed to stand without stirring. Samples were taken from the enzyme solutions in multiple incubation time points and the rate of enzyme deactivation was monitored and plotted in the graph shown in FIG. 4.
As shown in FIG. 4, extent of enzyme deactivation was significantly higher when the enzyme concentration was 0.6 g/L (see open triangle symbols) compared to the case where the concentration was 6 g/L (see open circle symbols). Also, it was demonstrated that the rate of enzyme deactivation was higher when the solutions were stirred compared to the cases in which the solutions were allowed to stand (compare open and solid circles, or open and solid triangles).
In previous researches and developments investigating speed of stirring and a structure of stirring apparatus, experiments have been performed in a condition where the concentration of the saccharifying enzyme is high. Therefore, they have been performed in a condition in which the rate of enzyme deactivation due to a physical stress, such as stirring, is inherently low. When the concentration of the saccharifying enzyme is reduced to decrease the amount of the enzyme used, the enzyme is deactivated at an even higher degree. Accordingly, the lower the enzyme concentration, the higher the rate of the enzyme deactivation.
Because of reasons described above, the rate of enzyme deactivation becomes higher under a condition where the concentration of the enzyme is low and the mixed solution is stirred or shaken, reducing the degradation rate of cellulose and/or hemicellulose. Because of the lack of knowledge of the enzyme deactivating effect under the condition where the enzyme concentration is low and the mixed solution is subjected to physical stress, reducing the amount of the saccharifying enzyme used without a significant ratio of the enzyme being deactivated has been practically impossible.
To solve the problem described above, in the method described in Patent Literature 1, the enzymatic reaction is proceeded in a condition where the mixed solution is allowed to stand without stirring or shaking, contrary to the conventional methods, in which the mixed solution is stirred or shaken. By adopting the method described in Patent Literature 1, the amount of enzyme used has been reduced.
An disadvantage of the method described in Patent Literature 1 is that obtaining evenly distributed temperature and ingredients in the mixed solution becomes difficult without stirring or shaking when the reaction apparatus is scaled up in practical use. In short, the method described in Patent Literature 1 has an advantage and disadvantage. The advantage is that the rate of enzyme deactivation is lowered even if the concentration of the enzyme was low. The disadvantage is that the enzymatic reaction proceeds inefficiently because of the unevenly distributed temperature and ingredient in the mixed solution.